1. Technical Field
The subject invention relates to the cloning, expression and isolation of recombinant forms of amyloid beta protein containing a N-terminal methionine (and/or one or more other amino acid residues) as well as to methods of using these recombinant proteins.
2. Background Information
Amyloid β(1-42) protein, also referred to as Aβ(1-42), is a family of proteins ranging from 1-39, 1-40, 1-41, 1-42 and 1-43 residues in length. The 1-42 form is a central component of insoluble extracellular depositions (senile or neuritic plaques) composed of proteins, lipids, carbohydrates and salts in the brains of Alzheimer and Down's syndrome patients (Masters et al., PNAS 82, 4245-4249, 1985). In particular, amyloid β(1-42) and similar derivatives) protein is a polypeptide having 42 amino acids which is derived from the amyloid precursor protein (APP) by proteolytic processing. This also includes, in addition to human variants, isoforms of the amyloid β(1-42) protein present in organisms other than humans, in particular, other mammals, especially rats. This protein, which tends to polymerize in an aqueous environment, may be present in very different molecular forms.
A simple correlation of the deposition of insoluble protein with the occurrence or progression of dementia disorders such as, for example, Alzheimer's disease has proved to be unconvincing (Terry et al., Ann. Neurol. 30. 572-580 (1991), Dickson et al., Neurobiol. Aging 16, 285-298 (1995)). In contrast, the loss of synapses and cognitive perception seems to correlate better with soluble forms of Aβ(1-42)(Lue et al., Am. J. Pathol. 155, 853-862 (1999); McLean et al., Ann. Neurol. 46, 860-866 (1999)).
With the soluble forms of Aβ(1-42), there are essentially two different hypotheses regarding the to molecular forms that supposedly cause dementia disorders such as Alzheimer's disease. Firstly, a cytotoxic action of Aβ(1-42) protofibrils is postulated. The latter are still soluble, fibrillar, relatively highly aggregated Aβ(1-42) forms having molecular weights in the range from 150-250 kDa (Arispe et al. PNAS 90, 567 (1993), Lashuel et al., Nature 418, 291 (2002)) which, due to pore-forming properties, apparently cause an uncontrolled calcium influx through the membranes of neuronal cells. Secondly, oligomeric Aβ(1-42) derivatives having molecular weights in the range from 15-30 kDa have been described (M. P. Lambert et al. PNAS 95, 6448-6453 (1998)). These nonfibrillar oligomers also referred to as amyloid derived, diffusible and dementing ligands or ADDL's (see U.S. Pat. No. 6,218,506; International Application No. WO 01/10900; and Lambert et al., supra) can be found in preparations showing an inhibiting influence on the rate of long-term potentiation of neurons in hippocampal sections. However, the state of previous research on oligomers is characterized by great uncertainty over the actually relevant species. The information in the literature differs greatly. For example, U.S. Pat. No. 6,218,506 describes ADDLs having from 3 to 12 subunits, whereas the ADDLs described in International Application No. WO 01/10900 may have up to 24 subunits (see also Luhrs et al., PNAS 102(48), 17342-17347 (2005)).
In addition, the occurrence of N-terminally truncated forms of the Aβ(1-42) protein in connection with Alzheimer's disease has been reported Apart from Aβ(1-42), N-terminally truncated forms were also detected in the depositions of brains of deceased Alzheimer patients as early as 1985 (C. Masters et al., PNAS 92, 4245-4249 (1985)). Thus, particular proteases present in the brain, such as neprilysin (NEP 24.11) or IDE (short for insulin degrading enzyme), are also known to be able to degrade Aβ(1-42) (D. J. Selkoe, Neuron 32, 177-180, (2001)). However, the importance of the N-terminally truncated forms in the pathogenesis of Alzheimer's disease is unclear (Lee et al, JBS 278, 4458-4466 (2003)). Interestingly, some patients suffering from sporadic or familial Alzheimer's disease or Down's syndrome preferentially accumulate these truncated forms (J. Näslund et al., PNAS 91, 8378-8382, (1994), C. Russo et al., Nature 405, 531-532, (2000), T. C. Saido et al, Neuron 14, 457-466, (1995)). A relatively recent study (Sergeant et al., J. of Neurochemistry 85, 1581-1591, (2003)) showed that 60% of all insoluble Aβ peptides in the brains of deceased Alzheimer patients are based on N-terminally truncated forms.
In view of the above, there is certainly a need to produce large quantities of amyloid beta protein (of various forms) in order to meet the requirements of studies and resulting assays related to the detection of Alzheimer's Disease in humans and, more importantly, in the development of biologics and small molecules for the prevention and treatment of Alzheimer's Disease. Further, such production allows for further study into the properties of the protein in connection with the etiology of Alzheimer's. Thus, methods for producing this protein in sufficient quantity and in a native-like, functional form are invaluable.
Additionally, previous studies in vitro have relied upon chemically synthesized sequences which would result from natural cleavage points of the peptide. Also, studies in vivo typically rely upon biologically synthesized amyloid beta which self-evidently results from cleavage at the natural cleavage points. Thus, having the ability to produce large quantities of native and mutant amyloid peptides in a prokaryotic recombinant system such as E. coli will certainly be preferable to production methods currently in existence.
All patents and publications referred to herein are hereby incorporated in their entirety by reference.